DNA-protective effects of quercetin or naringenin in alloxan-induced diabetic mice

European Journal of Pharmacology 656 (2011) 110–118

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Immunopharmacology and Inflammation

DNA-protective effects of quercetin or naringenin in alloxan-induced diabetic mice Nada Oršolić a,⁎, Goran Gajski b, Vera Garaj-Vrhovac b, Domagoj Đikić a, Zvjezdana Špacir Prskalo c, Damir Sirovina a a b c

Department of Animal Physiology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia Institute for Medical Research and Occupational Health, Mutagenesis Unit, HR-10000 Zagreb, Croatia Laboratory of Clinical Chemistry, University Hospital for Tumours, Ilica 197, HR-10000 Zagreb, Croatia

a r t i c l e

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Article history: Received 17 July 2010 Received in revised form 15 December 2010 Accepted 7 January 2011 Available online 28 January 2011 Keywords: Quercetin Naringenin Alloxan Diabetes DNA damage (Mouse)

a b s t r a c t Diabetes mellitus is associated with a high production of reactive oxygen species, which may cause oxidative DNA damage. High levels of genomic damage have been associated with liver and renal failure as well as immune-system decline. Flavonoids are effective antioxidants and may protect against several chronic diseases including diabetes. This study used the comet assay to assess the levels of DNA damage in the blood, liver and kidney cells in untreated and quercetin (QU) or naringenin treated diabetic mice. In addition, the study was designed to establish whether QU or naringenin might have a biological effect in protecting diabetic mice against oxidative stress by using survival studies to observe total body injury at the level of the organism. QU or naringenin were injected to mice intraperitoneally (i.p.) at a dose of 50 mg/kg for 7 days starting 2 days after a single dose (75 mg/kg, i.v.) alloxan injection. These findings suggest that QU or naringenin treatment resulted in a significant increase in the body weight, the haematological and immunological parameters of blood, as well as leading to 100% survival of diabetic mice. The tested flavonoids have protective effects against alloxan-induced DNA-damage in peripheral lymphocytes but not in the liver and kidney cells of diabetic mice. It might be hypothesised that diabetic mice with a high intake of flavonoid-rich foods, and specifically foods rich in quercetin or naringenin, might be relatively protected against long-term complications of diabetes due to decreased oxidative stress. Various co-operative and synergistic action mechanisms of the tested flavonoids may lead to the protection of the whole organism against diabetes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Diabetes mellitus is possibly the world's fastest growing metabolic disorder, and as the knowledge of the heterogeneity of this disorder increases, so does the need for more appropriate therapy (World Health Organization, 2002; Oršolić and Bašić, 2008). Diabetes mellitus is a pathologic condition, resulting in severe metabolic imbalances and non-physiologic changes in many tissues, where oxidative stress plays an important role in the aetiology (Yue et al., 2003; Oršolić and Bašić, 2008). Diabetes is associated with the generation of reactive oxygen species (ROS), which cause oxidative damage, particularly to heart, kidney, eyes, nerves, liver, small and large blood vessels, immunological and gastrointestinal system (Obrosova et al., 2003a,b; Yue et al., 2003; Oršolić and Bašić, 2008). Diabetics and experimental animal models exhibit high oxidative stress due to persistent and chronic hyperglycemia, thereby depleting the activity of the antioxidative defence system and promoting free radical generation (Ihara et al., 1999; Nishikawa et al., 2000). Such Abbreviations: NMP, normal melting point agarose; LMP, low melting point agarose. ⁎ Corresponding author at: Department of Animal Physiology, University of Zagreb, Rooseveltov trg 6, 10 000 Zagreb, Croatia. Tel.: +385 1 4877 735; fax: +385 1 4826260. E-mail address: [email protected] (N. Oršolić). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.021

models include alloxan or streptozotocin induced diabetic rats and mice (Lenzen and Panten, 1988; Sharmna and Garg, 2009; Venkatesh et al., 2010). In alloxan induced type 1 diabetes, hyperglycemia and oxidative stress have been implicated in the aetiology and pathology of disease complications (Winterbourn and Munday, 1989; Szkudelski et al., 1998; Blasiak et al., 2003; Wu et al., 2010). The ROS and nitric oxide induced by cytokine may interact and modify cellular protein, lipid and DNA, which results in altered target cell function (Blasiak et al., 2003). Hyperglycaemia largely influences neutrophil and macrophage functions. In uncontrolled blood sugar levels, impairment in chemotaxis, phagocytosis and microbicidal functions has been demonstrated (Molenaar et al., 1976). Excessively high blood sugar levels, acidosis, ketosis and hyperlipidemia are presumed to be the responsible factors for disturbances in lymphocyte functions, which are suppressed in patients with an irregular course of illness (Jirkovská et al., 2002; Spatz et al., 2003). The enormous costs of modern medicines indicate that alternative strategies are required for better management of diabetes. Traditional plant medicines are used throughout the world for a range of diabetic complications (Malviya et al., 2010; Qi et al., 2010). The study of such medicines might offer opportunities for the future. There has been a growing interest in flavonoids, which are widely distributed in plants and ingested by humans, due to their antioxidative,

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mild estrogenic, hypolipidemic, antibacterial, antitumor, anti-inflammatory, antidiarrhoeal, antiulcer, antimutagenic, myocardial protecting, vasodilator, immunomodulator and hepatoprotective activities (Choi et al., 1991a,b; Bhathena and Velasquez, 2002; Oršolić and Bašić, 2008). Consumption of flavonoids, particularly the flavonol quercetin (3,5,7,3,4-pentahydroxy-flavone), has been associated with a reduced incidence of heart disease and cancer (Sies et al., 2005; Oršolić et al., 2005; Oršolić and Bašić, 2007; Lea et al., 2010), hypothesised to be due to their antioxidant properties. We have recently shown that flavonoids have very high antioxidant activities (Benković et al., 2008; Oršolić et al., 2008, 2010) and may protect normal tissue cells against ionising radiation. Among naturally occurring flavonoids, naringin has been empirically proven to have no side-effects, as humans have been ingesting grapes and citrus fruits for a long time (Choe et al., 2001).The citrus-derived flavonoid, naringenin, has both lipid-lowering and insulin-like properties. In streptozotocin-induced diabetic rats, a diet supplement of naringenin 7-O-D-glucoside reduces blood glucose and improves plasma lipids (Choi et al., 1991b). In cholesterol-fed rats, naringenin lowers plasma cholesterol by inhibiting hepatic cholesterol synthesis and esterification (Jeon et al., 2007). Furthermore, naringenin potentiates intracellular signalling responses to low insulin doses, suggesting that naringenin sensitises hepatocytes to insulin (Mulvihill et al., 2009). There is a considerable body of evidence indicating that early detection and prompt treatment of diabetes can delay the onset, and slow down or prevent the progression of complications associated with diabetes. Much interest has gathered in the role and usage of natural antioxidants as a means to prevent oxidative damage in diabetes with high oxidative stress. Several of these flavonoids, including silymarin, catechin, and querectin, have shown protective effects in experimental diabetes by enhancing the activity of antioxidant enzymes (Sabu et al., 2002; Anathan et al., 2003; Gupta et al., 2004). The present study was thus undertaken to assess the protective effect of QU or naringenin on oxidative damage induced by alloxan in mouse blood, liver and kidney tissue and their possible role in ameliorating the development of diabetes. In addition, we investigated the antidiabetic effect of QU or naringenin on molecular, cellular and organism level. The results of the study could serve as a step towards the development of a mechanism-based therapeutic approach for the management of diabetes and hence provide the basis for the usefulness of two potent antioxidants, QU and naringenin. 2. Material and methods 2.1. Animals Male and female CBA inbred mice 2 to 3 months old, weighing 20 to 25 g, obtained from Department of Animal Physiology, Faculty of Science, University of Zagreb, were used in this study. The animals were kept in individual cages during the experiment and at 12 h of light per day. They were fed a standard laboratory diet (4 RF 21, Mucedola, Settimo Milanese, Italy) and tap water ad libitum. Maintenance and care of all experimental animals were carried out according to the guidelines in force in Republic of Croatia (Law on the Welfare of Animals, N.N. #19, 1999) and carried out in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS Publ. # (NIH) 86-123. 2.2. Reagents The test strips of blood glucose (Betachek Visual blood glucose test strips, Sydney, Australia), the kits of total cholesterol and triglyceride (Cholesterol Reagent and Triglycerides Reagent (Thermo Electron, Australia) and Alloxan (Sigma-Aldrich Chemical Co., USA) were used in the study. Quercetin dehydrate (QU) and naringenin were

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purchased from Fluka, BioChemica, Switzerland and from Sigma, Germany, respectively. 2.3. Experimental design Seventy mice were randomly divided into four groups, as follows: Group (i): control animals (normal, nondiabetic animals); received 0.5 ml distilled water intraperitonealy (i.p.) per day by injection for 7 days; Group (ii): alloxan controls; injected i.v. with alloxan in a single dose of 75 mg/kg body weight; these served as the untreated diabetic group; Group (iii): received quercetin (QU) i.p. in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection; these served as the QU-treated diabetic group. Group (iv): received naringenin i.p. in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection; these served as the naringenin-treated diabetic group. Five mice from each group were used on the 9th day after alloxan injection. After disinfection of the external abdominal region, each animal was inoculated with 3 ml of saline solution and after gentle agitation of the abdominal well, the solution containing peritoneal cells was removed for cellular evaluation. The following variables were analysed: the total number of cells present in the peritoneal cavity, the functional activity of the macrophages, haematological and biochemical parameters, total cholesterol and triglyceride, micronucleus assay and comet assy. The remaining animals, i.e., 8–11 animals of each group were used for the survival analysis. 2.3.1. Induction of experimental diabetes and determination of serum glucose level Diabetes was induced in Swiss albino mice by a single intravenous injection of alloxan monohydrate (75 mg/kg, i.v.) in total volume of 0.5 ml of freshly prepared saline solution. Blood glucose level was tested before alloxan injection and 48 h after treatment, to monitor the immediate diabetogenesis. After 48 h, the animals with fasting blood glucose level above 11 mmol L− 1 were selected for the study (diabetic mice) (Lan et al., 2010) and then treated with QU or naringenin. Blood glucose level was determined by test strips of blood glucose (Betachek Visual blood glucose test strips, Sydney, Australia). 2.3.2. Effect of QU or naringenin on body weight in alloxan induced diabetic mice During the study period of 50 days, the body weights of the mice were recorded every 4 days using an electronic balance. From these data, the mean change in body weight was calculated. 2.3.3. Count of the total number of cells present in the peritoneal cavity The total number of cells present in the peritoneal cavity was determined by counting in a Bürker–Türk chamber. 2.3.4. Determination of functional activity of macrophages The functional activity of macrophages in the peritoneal cavity was determined by the spreading technique adapted from Rabinovitch and DeStefano (1973) Thus, 103 cells in 0.1 ml of the cellular suspension obtained from the peritoneal cavity were placed over glass cover slips at room temperature for 15 min. The non-adherent cells were removed by washing with phosphate buffered saline, and the adherent cells were incubated in culture medium 199 containing 10 nM HEPES at 37 °C for 1 h. Following this, the culture medium was removed and the cells were fixed with 2.5% glutaraldehyde. Then, the cells were stained with a 5% solution of Giemsa and examined by optical microscopy where the percentage of spread cells were determined using a ×40 objective. Spread cells were those that exhibited cytoplasmic elongation, while the non-spread cells were rounded (Rabinovitch and DeStefano, 1973).

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2.3.5. Survival analysis For the survival analysis Swiss albino mice were given test components i.p. at doses of 50 mg/kg for 7 days starting 2 days after the alloxan injection. The end point of the experiment was determined by the spontaneous death of animals. The results are expressed as percentage of mean survival time of the treated animals over the mean survival time of the control group with diabetes (treated vs. control, T/C %). The percentage of increased lifespan (ILS%) was calculated according the formula: ILS% = (T − C)/C × 100 where T represents mean survival time of treated animals and C represents mean survival time of the control group. 2.3.6. Haematological analysis The haematological analysis was performed on blood obtained from the tail vein of experimental and control mice on day 9 after alloxan injection. Blood was collected into EDTA tubes. The measurement of the leukocyte, erythrocytes, haemoglobin, hematocrit, MCV, MCH, MCHC and platelets was made in an automatic cell counter (Cell-Dyn® 3200, Abbott, USA). 2.3.7. Serum samples and biochemical determinations Animals were treated with test components, blood samples were collected and centrifuged at 2200 rpm for 10 min. Serum was used for the determination of total protein, glucose, urea, creatinine, bilirubin, aspartate and alanine aminotransferases (AST and ALT) and lactic dehydrogenase (LDH). Biochemical parameters were made using serum samples from both control and experimental groups in an automatic cell counter. Serum triglycerides and total cholesterol were determined by enzymatic methods according to the commercial kit's instructions (Thermo Electron, Australia). The total concentration of triglycerides or total cholesterol was estimated by measured the absorbance of sample and standard by spectrophotometer (Shimadzu, UV-160) at a wavelength of 500 nm. 2.3.8. The alkaline comet assay To analyse DNA damage after alloxan treatment and combined treatment with naringenin and quercetin, we used the comet assay under alkaline conditions, as described by Singh et al. (1988). Fully frosted slides were covered with 1% normal melting point (NMP) agarose (Sigma). After solidification, the gel was scraped off the slide. The slides were then coated with 0.6% NMP agarose. When this layer had solidified, a second layer, containing the whole blood sample mixed with 0.5% low melting point (LMP) agarose (Sigma) was placed on the slides. After 10 min. of solidification on ice, slides were covered with 0.5% LMP agarose. Slides were then immersed in freshly prepared ice-cold lysis solution (2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris–HCl, 1% sodium sarcosinate (Sigma), pH 10) with 1% Triton X-100 (Sigma) and 10% dimethyl sulfoxide (Kemika) for 1 h to lyse the cells and allow DNA unfolding. The slides were then placed on a horizontal gel electrophoresis tank, facing the anode. The unit was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM disodium EDTA, pH 13.0) and the slides were placed in this alkaline buffer for 20 min. to allow DNA to unwind and show alkali-labile sites. Denaturation and electrophoresis were performed at 4 °C under dim light. Electrophoresis was carried out at 25 V (300 mA) (20 min at 1 V cm− 1) for 20 min. After electrophoresis, the slides were rinsed gently three times with a neutralisation buffer (0.4 M Tris–HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide and covered with a coverslip. A total of one hundred randomly captured comets from each slide were examined using an epifluorescence microscope (Zeiss, Oberkochen, Germany) connected to an image analysis system (Comet Assay II; Perceptive Instruments Ltd, Haverhill, Suffolk, UK). To quantify DNA damage, the following comet parameters were evaluated: tail intensity (% of DNA) and tail moment. The occurrence of apoptotic and necrotic cells was also evaluated on the same slides using the alkaline comet assay. The

apoptotic and necrotic index was calculated as the percentage of cells with a highly spread tail and an undefined head of uniform intensity (indicating necrosis) and cells with diffuse fan-like tails and small heads (indicating apoptosis, Olive et al., 1993). 2.3.9. Peripheral blood micronucleus assay The peripheral blood smear was prepared as described by Holden et al. (1997). The blood was collected from the tail tip and a smear was prepared on pre-cleaned slides. The smears were allowed to dry at room temperature and fixed in absolute methanol for 5 min. After fixation, slides were stained with acridine orange (AO) and washed twice with phosphate buffer (pH 6.8) as described by Hayashi et al. (1994). 2.4. Statistical analysis The parameters of tail intensity and tail moment were evaluated using Statistica 5.0 package (StaSoft, Tulsa, OK). Each sample was characterised for the extent of DNA damage by considering the mean± S.E.M. (standard error of the mean). In order to normalise the distribution and to equalise the variances, a logarithmic transformation was applied to the data. Multiple comparisons between groups were done by means of ANOVA on log-transformed data. Post-hoc analysis of differences was done by the Scheffé test. Other results were expressed as means ±S.D. obtained from 2 experiments. The significance of the differences between the group means was tested by Student's t-test. The statistical significance of control and experimental groups in the micronucleus assay was evaluated by the χ2 test. The level of statistical significance was set at Pb 0.05. 3. Results 3.1. Effect of alloxan on blood glucose level The blood glucose level was strongly elevated on the second day after treatment and the average levels of blood glucose in each group of mice ranging between 20 and 30 mmol/l (data not shown). Alloxan at a dose of 75 mg/kg of body weight successfully caused diabetes in mice. The diabetic animals showed the following signs of the condition: polydipsia (abnormal thirst), polyuria (increased urine volume), weight loss (duo to lean mass loss), asthenia, and dehydration (due to the animal body's attempt to get rid of the excess blood glucose as the normal process of storing glucose in the body cells is impaired). 3.2. Effect of QU or naringenin on the survival of alloxan-induced diabetic mice The toxicity of alloxan was studied by observing of body weight and life span of animals with diabetes. The effects of QU or naringenin on the body weight of mice with alloxan-induced diabetes are shown in Fig. 1 and Table 1. The body weight was rapidly reduced in animals treated with alloxan alone; the fall was the largest between 3 and 10 days, and then body weight started to gradually recover, especially in diabetic animals treated with QU and/or naringenin, which had almost reached the mass of healthy (nondiabetic) animals. During the experiments, 50% of animals from the untreated group of mice with diabetes died, while animals treated with test components all survived, as shown in Table 1. 3.3. Effect of QU or naringenin on haematological and biochemical parameters Tables 2 and 3 show haematological and biochemical parameters in normal (nondiabetic) animals and experimental animals in each group. The number of leukocytes in diabetic mice treated with QU or

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3.4. Effect of QU and naringenin on the total number of peritoneal cells and the functional activity of macrophages in alloxan-induced diabetic mice The results (Fig. 2) showed a statistically significant difference in the total number of cells in the peritoneal cavity of diabetic mice treated with naringenin or QU compared to untreated diabetic animals (P b 0.05; P b 0.01, respectively) and nondiabetic animals. The results obtained in Fig. 3 indicated a statistically significant increase in the number of activated macrophages in untreated diabetic mice and diabetic mice treated with QU (P b 0.05) versus nondiabetic animals. Diabetic animals treated with QU (P b 0.001) showed a significantly increased number of functionally activated macrophages as compared with untreated diabetic mice (Fig. 3). 3.5. The alkaline comet assay

Fig. 1. Effect of naringenin or quercetin on the body weight of alloxan-induced diabetic mice. Control group of nondiabetic mice received 0.5 ml distilled water intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. Diabetic mice injected i.v. with alloxan in a single dose of 75 mg/kg body weight served as untreated diabetic group. Untreated diabetic mice received 0.5 ml 0.5% etOH (v/v) intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. Diabetic mice treated with naringenin or quercetin i.p. in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection.

naringenin was increased but without significant statistical difference as compared to untreated diabetic mice or nondiabetic mice (Table 2). Diabetic animals treated with naringenin had a reduced number of erythrocytes and haemoglobin (P b 0.01) as compared to control or untreated diabetic mice. No differences were noted in the other haematological parameters. As shown in Table 3, alloxan elevated the values of ALKP; a statistically significant difference existed between QU or naringenin (P b 0.01) treated diabetic mice and nondiabetic animals. A statistically significant increase in levels of urea and triglycerides was noted in diabetic groups (P b 0.05) compared to healthy (nondiabetic) animals or diabetic animals treated with tested flavonoids. Other biochemical parameters did not show any variability (Table 3).

The measured parameters of the alkaline comet assay measured in lymphocytes, liver and kidney cells are presented in Tables 4–6. Statistical analysis showed that alloxan treatment measured in lymphocytes increased DNA damage compared to the control (nondiabetic) animals whereas both naringenin and quercetin decreased DNA damage compared to the alloxan treatment. However, in liver and kidney cells alloxan treatment did not increased DNA damage compared to control animals. In both types of cells naringenin also did not affect DNA damage, while quercetin significantly increased both comet parameters compared to the control animals and to those treated with alloxan (P b 0.05). Apoptotic frequency in liver cells was not affected by the addition of naringenin and quercetin, while necrotic frequency was mildly increased. However, in kidney cells naringenin slightly increased both types of cell death whereas quercetin had a large impact on both (Fig. 4). 3.6. Micronucleus assay As shown in Table 7, the treatment of diabetic mice with tested flavonoids did not induce significant changes in the number of micronucleus in reticulocytes of peripheral blood as compared with nondiabetic and diabetic mice. However, the number of micronucleated cells in untreated diabetic mice was lower than in nondiabetic mice. 4. Discussion

Table 1 Survival of untreated diabetic mice and diabetic mice treated with naringenin or quercetin. Experimental group

Range of survival Median (days) ± SD

ILS%a

Diabetic mice + 0.5% etOHd Diabetic mice + naringenine Diabetic mice + quercetine

22–45

42 ± 9.68

–

45

45f

16.67 120

8

45

f

16.67 120

8

45

T/C% b

Long-term survivors (LTS)c

–

4

Results are expressed as the means of each group (n = 8) of and are representative of two independent experiments. Statistically significant difference between naringenin or quercetin treated diabetic mice and untreated diabetic animals (fP b 0.05; Log-Rank test). a ILS% (increased life span %) = (T − C)/C × 100. T, mean survival time of treated diabetic group; C, mean survival time of untreated diabetic group. b T/C treated diabetic animals versus untreated diabetic animals. c LTS — Long-term survivors; mice surviving more than 45 days after alloxan treatment. d Diabetic mice: injected i.v. with alloxan in a single dose of 75 mg/kg body weight and served as untreated diabetic group. Untreated diabetic mice received 0.5 ml 0.5% EtOH intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. e Diabetic mice treated with naringenin or quercetin i.p. in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection.

Over the years, various medicinal plants and their extracts rich in polyphenolic/flavonoid compounds have been reported to be effective against ROS mediated damage by enhancing antioxidants and reduces hyperglycemia in alloxan- or streptozotocin-induced diabetes. The hypoglycaemic activities of some of these phytochemical constituents have been evaluated and confirmed in animal models (Ugochukwu and Babady, 2003; Eddouks and Maghrani, 2004; Vinson and Zhang, 2005; Oršolić and Bašić, 2008). In the present study, we examined the possible usefulness of the potent antioxidants QU or naringenin at the molecular, cellular and organism level in alloxan-induced diabetes. Flavonoids quercetin or naringenin, used in doses of 50 mg/kg body mass were shown to be capable of normalising body weight, serum cholesterol and triglyceride concentration, significantly reducing blood DNA damage and augmenting the life span of alloxandiabetic mice. A beneficial antidiabetic effect of QU or naringenin was noticed on the lifespan of diabetic mice in our study (Table 1). Out of 8 alloxanized animals, 4 died within 22 days in the control diabetic group versus a 100% survival rate of mice in the diabetic groups treated with QU or naringenin. On the other hand, our data showed that treatment of diabetic mice with QU or naringenin resulted in marked increase in animal body weight (Fig. 1). Body weight was rapidly reduced in animals treated with alloxan alone; the fall was the largest between 3

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Table 2 Haematological parameters of nondiabetic mice, untreated diabetic mice and diabetic mice treated with naringenin or quercetin.

WBC(× 109/l) Mononuclear/% Polymorphonuclear/% RBC(× 1012/l) HGB (g/l) HCT (l/l) MCV (fl) MCH (pg) MCHC (g/l) RDW/%CV PLT (×109/l) MPV(fl)

Nondiabetic micea

Diabetic mice + 0.5% etOHb

Diabetic mice + naringeninc

Diabetic mice + quercetinc

4.85 ± 1.01 57.06 ± 12.081 42.94 ± 12.08 9.13 ± 0.32 135.55 ± 5.35 0.444 ± 0.024 97.2 ± 1.91 29.7 ± 0.26 616.7 ± 6.43 24 ± 3.01 1434.5 ± 373.75 9.76 ± 0.67

4.41 ± 1.31 51.73 ± 12.62 48.27 ± 12.62 9.435 ± 0.278 136.2 ± 3.67 0.452 ± 0.011 95.8 ± 1.50 28.9 ± 0.41 603 ± 7.74 23.6 ± 1.78 1521.5 ± 251.01 9.92 ± 0.63

5.57 ± 3.19 44.28 ± 13.73 55.725 ± 13.726 8.765 ± 0.1e 126.05 ± 4.024ef 0.423 ± 0.01e 96.5 ± 1.43 28.8 ± 0.71 595.5 ± 6.60 22.6 ± 1.96 1201 ± 512.77 10.95 ± 0.54df

9.05 ± 8.24 52.93 ± 13.94 47.07 ± 13.94 7.805 ± 2.01 114.9 ± 31.75 0.379 ± 0.093 97.2 ± 2.78 29.35 ± 2.23 602.5 ± 35.49 27.1 ± 5.39 1309 ± 266.66 9.95 ± 1.02

Statistically significant difference between naringenin or quercetin treated diabetic mice and untreated diabetic animals (dP b 0.05; eP b 0.01; Studentov t-test). Statistically significant difference between nondiabetic animals and naringenin or quercetin treated diabetic mice (fP b 0.05; Student's t-test). a Control group of nondiabetic mice received 0.5 ml distilled water intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. b Diabetic mice; injected i.v. with alloxan in a single dose of 75 mg/kg body weight; served as untreated diabetic group. Untreated diabetic mice received 0.5 ml 0.5% etOH intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. c Diabetic mice treated with naringenin or quercetin ip in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection.

and 10 days, and then body weight started to recover easily. In diabetic animals treated with QU and/or naringenin, the body weight was only slightly reduced and the treated mice almost reached the mass of healthy (nondiabetic) animals. The improved body weight of diabetic mice treated with QU or naringenin could be due to a better control of the hyperglycaemic state in the diabetic mice. It is likely that decreased body weight in diabetic animals is due to dehydration and catabolism of fats and proteins (Hakim et al., 1997). Increased catabolic reactions leading to muscle wasting might also be the reason for the reduced weight gain by diabetic animals (Rajkumar et al., 1991). The findings of our study are in agreement with the findings of Choi et al. (1991a,b), Choe et al. (2001) and Jeon et al. (2007) and show that QU or naringenin reduce the total level of cholesterol and triglycerides in the blood of a diabetic mouse, which are one of the major risk factors of cardiovascular disease. So, QU or naringenin could modulate lipid metabolism (Table 3) and reduce the syndrome caused by blood lipid abnormalities (Fuliang et al., 2005). Thus, both comet assay parameters revealed that alloxan treatment induced DNA damage in the lymphocytes of treated animals, whereas naringenin and quercetin proved themselves as potent protectors against alloxan-induced DNA damage, which was indicative from the decrease of DNA migration compared to the alloxan treatment (Table 4). On the contrary, in liver and kidney cells we observed a different outline of action (Tables 5 and 6). Alloxan in those cells failed to significantly increase DNA damage compared to the unexposed mice. Also, naringin in those cells had no impact on DNA migration, whereas quercetin in both types of cells significantly

damaged DNA molecules compared to the untreated animals and those treated with alloxan. Furthermore, the comet assay was used to measure apoptotic and necrotic cell death based on the cell appearance on microscope slides. While no increase of either apoptosis or necrosis was determined in peripheral lymphocytes, the liver and kidney cells both showed a similar pattern of incremental apoptotic and necrotic cell death (Fig. 4). In liver cells there were no changes in the percentage of apoptosis, while necrotitic cell death was slightly elevated after treatment with naringenin and quercetin. In kidney cells, however, naringenin produced a slight increment, whereas quercetin strongly affected the percentage of both types of cell death, putting it above 100. Increased DNA damage, determined here, may derived from elevated ROS production via metal-catalysed auto-oxidation of glucose and/or autooxidative acitivity of QU. It is known that during its antioxidative activities, quercetin becomes oxidised into various oxidation products (Boots et al., 2008; Oršolić et al., 2010). It is possible that autooxidative activity of quercetin may be the result of a high concentration of quercetin and its inhibition of mitochondrial respiration with concomitant production of the superoxide anion and hydrogen peroxide. Hydrogen peroxide causes DNA strand breakage by generation of hydroxyl radicals close to the DNA molecule (the Fenton reaction). Reactive oxygen species can attack all types of macromolecules including DNA. DNA damage analysis under alkaline conditions, as introduced by Singh et al. (1988), allows for the identification of single-and double-strand breaks of alkali-labile sites, characterised as points of cleavage in the polynucleotide chain during

Table 3 Biochemical parameters of nondiabetic mice, untreated diabetic mice and diabetic mice treated with naringenin or quercetin. Nondiabetic micea ALKP (U/l) AST (U/l) ALT (U/l) LD (g/l) TP (g/l) GLU (mmol/l) UREA (mmol/l) Cholesterol (mmol/l) Triglycerides (mmol/l)

53.75 ± 7.5 186.25 ± 31.72 66.25 ± 11.09 2632.5 ± 298.48 48.5 ± 4.38 9.89 ± 0.64 7.5 ± 1.22 3.52 ± 0.26 2.72 ± 0.16

Diabetic mice + 0.5% etOHb f

95 ± 10.80 183.75 ± 41.70 57.5 ± 19.36 2487.5 ± 253.42 48.75 ± 4.92 25.575 ± 4.55e 9.125 ± 0.42 3.20 ± 0.80 3.37 ± 1.11

Diabetic mice + naringeninc d

58.75 ± 11.08 155 ± 24.15 58.75 ± 14.36 2387.5 ± 361.58 43.125 ± 1.25 39.637 ± 1.92 g 8.875 ± 2.05 3.04 ± 0.51 2.22 ± 1.01

Diabetic mice + quercetinc 57.5 ± 15.23 283.75 ± 46.43d g 48.75 ± 13.14 3151.25 ± 929.27 44.5 ± 7.44 21.8375 ± 5.45 7.375 ± 0.23d 2.34 ± 0.42 1.50 ± 0.39d

Statistically significant difference between naringenin or quercetin treated diabetic mice and untreated diabetic animals (dP b 0.05; Student's t-test). Statistically significant difference between untreated diabetic mice and nondiabetic animals (eP b 0.05; fP b 0.01; Student's t-test). Statistically significant difference between nondiabetic animals and naringenin or quercetin treated diabetic mice (gP b 0.05; Student's t-test). a Control group of nondiabetic mice, received 0.5 ml distilled water intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. b Diabetic mice; injected i.v. with alloxan in a single dose of 75 mg/kg body weight; served as untreated diabetic group. Untreated diabetic mice received 0.5 ml 0.5% etOH intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. c Diabetic mice treated with naringenin or quercetin i.p. in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection.

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Table 4 Comet assay parameters (tail intensity and tail moment) in mouse lymphocytes after alloxan treatment and joint treatment with alloxan and naringenin or quercetin. Sample

Comet parameters Tail intensity

Control Alloxan Naringenin Quercetin

Tail moment

Mean ± S.E.M.

Median

Mean ± S.E.M.

Median

2.35 ± 0.13 3.46 ± 0.16a 2.71 ± 0.12b 2.71 ± 0.12 b

1.53 2.48 2.03 2.07

0.28 ± 0.02 0.43 ± 0.02 0.35 ± 0.02 0.34 ± 0.02

0.18 0.30 0.27 0.25

a b b

a

Statistically significant increase compared to control ( P b 0.05). Statistically significant decrease compared to alloxan treatment (bP b 0.05). Fig. 2. The total number of cells in the peritoneal cavity of nondiabetic mice (control), untreated diabetic mice (Alloxan) and diabetic mice treated with naringenin or quercetin. #Statistically significant difference between nondiabetic animals and untreated diabetic animals or diabetic animals treated with naringenin or quercetin (#P b 0.05; ##P b 0.01; Student's t-test).

DNA incubation under alkaline conditions. In the comet assay, damage level is proportional to the size of the comet's tail, i.e. the tail represents the amount of damaged genetic material that has migrated from the nucleus (Ostling and Johanson, 1984). According to our data (Fig. 2), it is likely that ROS react with plasma lipids leading to the generation of chemotactic factors, which are in turn capable of stimulating neutrophiles with subsequent release of enzymes stored in cytoplasmic granules and the additional production of reactive oxygen species (Zozulińska et al., 1996). Significantly increased number of cells in the abdominal cavity (Fig. 2) was mostly due to the increased the number of neutrophiles and other inflammatory cells. Analysis of the functional ability of macrophages (macrophage spreading) from the inflammatory infiltrate showed an increased activity of macrophages in animals with diabetes treated with QU or naringenin. QU or naringenin in therapeutic treatment increased the macrophage spreading activity (Fig. 3). It is likely that macrophage activation and their effects on other immunological effectors could be responsible for the reduction of infection caused by uncontrolled diabetes and for the long-term survival of animals. Activated macrophages were shown to be a major component of host defence against bacterial, viral and fungal infection as well as neoplastic growth in experimental tumour systems (Oršolić and

Bašić, 2007, 2008). Antioxidant ability of tested flavonoids can not only increase macrophage activity, but may be crucial in preserving other immune cells and preventing the weakening of the host immune reactivity (Table 2). In the literature, interest in the role and usage of natural antioxidants for preventing oxidative damage in diabetes has recently grown (Okutan et al., 2005; El-Alfy et al., 2005; Kao et al., 2006; Oršolić and Bašić, 2008). But no molecular, biochemical or immunological data are available about the effects of QU or naringenin on kidney and liver cells of diabetic animals. In the present study, QU treatment caused a sharp increase in the cell death of kidney cells in alloxan-induced diabetic mice. In this work, the evidence that QU or naringenin does not reduced functional capacity of kidneys (Table 3) came from urea levels. Urea concentration in the blood is a consequence of its production during amino acid catabolism and its excretion by the kidneys. Kidney damage is increased in alloxantreated mice. The activities of liver marker enzymes and their variation reflect the overall change in metabolism that occurs during malignancy or organ dysfunction (Oršolić et al., 2010). These enzymes have clinical importance. The activity of these enzymes can be used for diagnosis as indicators of the prognosis of disease as well as a potential molecular biomarker for assessing exposure to any toxic agents. Tissue damage is the sensitive feature, so any deterioration or destruction of the membrane can lead to the leakage of these enzymes from the tissues. These enzymes are widely distributed in tissues: AST is predominantly found in the heart, liver, skeletal muscle, kidney and pancreas; ALT in the liver, kidney and heart. LDH isoenzymes are used to diagnose specific organ injury, mainly in cardiatic damage, and are important as an index of cell viability. The rise in the AST level in QUtreated diabetic mice indicates that all those organs were affected at the level of the plasma membrane, where the lipid bilayer was distressed enough to release higher amounts of this transport enzyme in the blood. It is possible that QU has pro-oxidative effects in the presence of in vivo transition metal ions such as Fe2+ or Cu+, which catalyse the conversion of the superoxide anion and hydrogen peroxide into the highly reactive hydroxyl radical, which in turn provokes a broad spectrum of DNA lesions (Aruoma et al., 1996). It seems that the increase of LDH and AST are due to hepatotoxic

Table 5 Comet assay parameters (tail intensity and tail moment) in mouse liver cells after alloxan treatment and joint treatment with alloxan naringenin or quercetin. Sample

Comet parameters Tail intensity

Fig. 3. The percentage of macrophage spreading in the peritoneal cavity of nondiabetic mice (control), untreated diabetic mice (Alloxan) and diabetic mice treated with naringenin or quercetin. Mice (n = 8) of each group were sacrificed on the 9th day after i.v. alloxan injection. Columns are expressed as mean ± standard deviation (SD). *Statistically significant difference between diabetic mice treated with quercetin and untreated diabetic animals ( ***P b 0.001; Student's t-test). #Statistically significant difference between nondiabetic animals and untreated diabetic animals or diabetic animals treated with quercetin (#P b 0.05; Student's t-test).

Control Alloxan Naringenin Quercetin

Tail moment

Mean ± S.E.M.

Median

Mean ± S.E.M.

Median

0.88 ± 0.08 1.08 ± 0.14 1.02 ± 0.12 6.66 ± 0.38ab

0.57 0.41 0.38 5.13

0.10 ± 0.01 0.14 ± 0.02 0.14 ± 0.02 1.06 ± 0.06ab

0.07 0.05 0.05 0.80

Statistically significant increase compared to control (aP b 0.05). Statistically significant increase compared to alloxan treatment (bP b 0.05).

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Table 6 Comet assay parameters (tail intensity and tail moment) in mouse kidney cells after alloxan treatment and joint treatment with alloxan and naringenin or quercetin. Sample

Comet parameters Tail intensity

Control Alloxan Naringenin Quercetin

Tail moment

Mean ± S.E.M.

Median

Mean ± S.E.M.

Median

1.32 ± 0.14 1.32 ± 0.32 1.83 ± 0.23 4.14 ± 0.84ab

0.63 0.54 1.07 4.07

0.16 ± 0.02 0.17 ± 0.04 0.23 ± 0.03 0.59 ± 0.12ab

0.07 0.07 0.13 0.58

Statistically significant increase compared to control (aP b 0.05). Statistically significant increase compared to alloxan treatment (bP b 0.05).

damage. LDH is also specific to muscle cells but could also point to hepatic damage. It is possible that the high concentration of QU in direct contact with kidney cells or liver cells during 7 days might cause increased damage to these cells in alloxan-induced diabetes, but we should point out that plasma levels of QU would be much lower following oral administration, especially due to its rapid conjugation with glucoronid acid and methylation in the body. When quercetin was administered orally, it was poorly absorbed from the digestive tract and did not have a great influence on the organs (Yoshida et al., 1990; Murota and Terao, 2003). However, the toxicity of alloxan was observed as histological changes in the kidney (Ashok et al., 2007); the shrinkage of glomeruli and the presence of phagocytes in tubulae was observed. In addition, it is possible that a lower percentage of apoptosis/necrosis in kidney or liver cells in alloxan-treated mice might be a consequence of the rapid destruction of liver or kidney cells and that QU or naringenin prolonged the life span of alloxandamaged cells. The reduced number of micronuclei in the untreated diabetic animals confirms our hypothesis. Numerous data showed that QU treatment protects the majority of the cells of the Langerhans islet and might increase the area of insulin immunoreactive β-cells significantly if QU was injected i.p. 3 days before streptozotocininduced diabetes in rats at dose of 15 mg/kg (Coskun et al., 2005). Hii and Howell (1984) reported that exposure of isolated rat islets to certain flavonoids such as (−)-epicatechin or quercetin enhances insulin release by 44–70%. They argue that such flavonoids may act on islet function, at least in part, via alteration in Ca2+ fluxes and in cyclic

Table 7 Number of reticulocytes with micronuclei in peripheral blood of nondiabetic mice, untreated diabetic mice and diabetic mice treated with naringenin or quercetin. Experimental group

Without MN ± S.D.

With MN ± S.D.

Nondiabetic micea Diabetic mice + 0.5% etOHb Diabetic mice + naringeninc Diabetic mice + quercetinc

1996.75 ± 0.5 1997.75 ± 0.5 1995.25 ± 2.36 1996.25 ± 3.3

3.25 ± 0.5 2.25 ± 0.5d 4.75 ± 2.36 3.75 ± 3.3

Statistically significant difference between untreated diabetic mice and nondiabetic animals (dP b 0.05; χ2 test). a Control group of nondiabetic mice received 0.5 ml distilled water intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. b Diabetic mice; injected i.v. with alloxan in a single dose of 75 mg/kg body weight; served as untreated diabetic group. Untreated diabetic mice received 0.5 ml 0.5% EtOH intraperitonealy (i.p.) for 7 consecutive days starting 2 days after alloxan injection. c Diabetic mice treated with naringenin or quercetin i.p. in a daily dose of 50 mg/kg for 7 days starting 2 days after alloxan injection.

nucleotide metabolism. Vessal et al. (2003) suggested that QU supplementation has proven to be beneficial in decreasing blood glucose concentration, promoting regeneration of the pancreatic islets and increasing insulin release in streptozotocin-induced diabetic rats, thus exerting its beneficial antidiabetic effects. The present investigation reports the protective effect of QU or naringenin on oxidative damage induced by alloxan in mouse blood, and their possible role in ameliorating the development of diabetes. Reduced oxidative stress and stronger immune capacity of the body are the likely reasons for the good condition of diabetic mice treated with QU or naringenin and for their overall survival. It is possible that flavonoids such as QU or naringenin cause increased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH), (Molina et al., 2003; Oršolić and Bašić, 2005). This might lead to radical scavenging abilities, tissue regeneration properties, wound healing characteristic, immunostimulatory and anti-inflammatory effects, which may influence the survival of the diabetic mice. It is likely that various co-operative and synergistic mechanisms of the tested flavonoids contribute to the protection of the whole organism against alloxan-induced diabetes. As a conclusion, QU or naringenin show protective effects in experimental diabetes, possibly by decreasing oxidative stress by their antioxidant nature (Coskun et al., 2005; Oršolić and Bašić,

Fig. 4. Percentage of apoptotic and necrotic cell deaths counted visually during the alkaline comet assay in mouse liver (A) and kidney (B) cells after alloxan treatment and joint treatment with alloxan and naringenin or quercetin.

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